Telomeric DNA abnormalities are a critical and universal aspect of carcinogenesis. The gradual replicative loss of telomeres ultimately results in telomere dysfunction and plays a role in many age-related diseases, including cancer. Sporadic telomere deletion events are a universal cell-intrinsic mutational mechanism that can lead to dysfunctional telomeres and chromosome instability; the frequency of these events is believed to depend upon factors such as DNA replication fork stalling and other DNA replication stress, oxidative damage, and homologous recombination events such as unequal sister chromatid exchange (SCE) and T-loop recombination. Sporadic telomere deletion events occur at a very low frequency in normal cells; an increased frequency of these events (and hence dysfunctional telomeres) may be among the very first mutational events in carcinogenesis. Repair of dysfunctional telomeres can result in telomere-telomere fusions, telomere- chromosome arm translocations at sites of internal double-strand DNA breaks, and additional DNA rearrangements as a consequence of repeated fusion-breakage-fusion cycles that result in genome instability and help drive cancer progression. Eventually, activation of telomere maintenance mechanisms (either telomerase-based or ALT-based) are believed to help stabilize dysfunctional telomeres and permit rapid growth of tumor cells. It is impossible to measure accurately the global frequency of sporadic telomere deletion events and telomere fusions with current technology. As a consequence, telomere mutational data are totally absent from the high- throughput datasets being acquired from tumor samples as part of the Cancer Genome Atlas, and telomere mutational data gleaned from a few labor-intensive studies of telomere function in cellular and organismal cancer models are incomplete and biased. Our lab has focused upon detailed analyses of human telomeric DNA structure and variation; here, we propose to use this knowledge to develop a universal, high throughput assay for detection and quantitation of telomeric DNA mutational events in humans and to obtain proof-of-principle data for its utility. The method couples the physical enrichment and purification of telomeric DNA with quantitative analysis of the telomeric genome fraction by high-throughput paired-end sequencing. It is designed to detect and quantitate single-allele-resolution ultrashort (TTAGGG)n tract profiles, telomere fusions, and subterminal DNA breakage-rejoining events. In addition, it is amenable to future refinement to permit miniaturization and multiplexing of the assays. The quantitative, single-allele-resolution measurements of telomere length and instability will permit unprecedented insights into the role(s) telomere loss and telomere fusion play in carcinogenesis, including mechanistic insights into molecular events mediating these processes and translational insights for the potential prognostic and tumor stratification applicability of the method.